Methods for transferring foreign genes into host cells, that is, the transformation technique, are fundamental techniques in the field of gene engineering. Such methods are used to analyze functions of transferred genes, produce recombinant proteins, gene therapy, generate useful recombinant plants, etc.
Previously developed techniques for transforming plant cells include the electroporation method, the Agrobacterium-mediated method, the microprojectile bombardment method, and the polyethylene glycol method. In the electroporation method, a DNA is transferred using an electric pulse. The Agrobacterium-mediated method utilizes infection by a terrestrial bacterium, Agrobacterium, which causes a plant tumor. In the microprojectile bombardment method, microparticles adsorbing a DNA are shot into cells. The polyethylene glycol method comprises binding a DNA to polyethylene glycol that is a fusion agent and transferring the binding product into cells by (presumably) an endocytosis-like process.
However, these methods cannot introduce macromolecular DNA fragments larger than 25 kb into cells (Carol, M. et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9975 (1996)). For example, it was difficult to apply the above-described transformation methods to isolate and clone genes of a map-base using a gene map made utilizing a restriction fragment length polymorphis (RFLP) marker. In order to transfer a long-chain DNA into cells, attempts were made to improve the above-described transformation methods. Recently, a long-chain DNA was successfully transferred into plant cells by the Agrobacterium-mediated method and the microprojectile bombardment method [Carol, M. et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9975-9979, (1996), Joyce, M. et al., Plant Cell Reports 14, 299-304 (1995)].
However, the Agrobacterium-mediated method has problems in that a foreign gene to be transferred is inserted into a limited vector system and this method cannot be applied to some plant species. The microprojectile bombardment method has disadvantages in that the apparatus used is expensive and the transfer efficiency is low.
In contrast, the polyethylene glycol method is highly applicable since this method can be performed simply if using a purified DNA and can be applied to an unlimited variety of plant cells. However, the transfer of a long-chain DNA into cells by this method has never been reported.